This application is generally in the field of drugs to treat drug resistant pathogens, and in particular relates to protease inhibitors that do not elicit drug-resistant mutations in the pathogens they inhibit, such as the human immunodeficiency virus (HIV).
Drug resistance generally is a problem with the treatment of most pathogens, including bacteria and viruses. A variety of methods have been used, the most common being determining which drugs the pathogen is sensitive to, then treating the patient with a drug that the pathogen is sensitive to. Another approach is the use of a “cocktail”, a mixture of two or three different drugs, preferably operating by different mechanisms of action, to block the life cycle of the pathogen before it can develop drug resistance. In the case of a virus such as HIV, this latter approach has been widely adopted, primarily through the use of one or two nucleoside drugs that inhibit replication by interacalation into the viral nucleic acid, in combination with a protease inhibitor that prevents replication. Unfortunately, even with the use of cocktails, HIV mutates extremely rapidly, and becomes resistant even to these combinations of drugs.
The HIV protease gene codes for a protease which, upon-expression as part of the gag-pol protein, procsses gag and gag-pol polyproteins into individual structural proteins and enzymes for the assembly of HIV virions (Debouck et al. (1987), J. Med. Chem. Res. 21:1-17). Mutation of the active-site residues of HIVPr renders the mutant virus non-infectious (Kohl et al. (1988), Proc. Natl. Acad. Sci. USA 85:4686-4690; Peng et al. (1989), J. Virol. 63:2550-2555), which established the HIVPr as a therapeutic target. As a result, many HIVPr inhibitors have been synthesized and tested (Wlodawer and Erickson (1993), Ann. Rev. Biochem. 62:843-855), among which four have been marketed: saquinavir (Ro 31-8959, Craig et al. (1991), Antiviral Res. 16:295-305), indinavir (L-735,524, Dorsey et al. (1994), J. Med. Chem. 37:3443-3451), ritonavir (ABT-538, Kempf et al. (1995), Proc. Natl. Acad. Sci. USA 92:2484-2488) and nelfinevir (Patick et al. (1996), Antimicrob. Agents Chemother. 40:292-297). Structures are shown in FIGS. 1a-d. These drugs are among the most powerful compounds to suppress HIV replication, as demonstrated both in tissue culture and in clinical trials (Wei et al. (1995), Nature 373:117-122; Ho et al. (1995), Nature 373:123-126). Combination therapies including HIVPr inhibitors have offered the best results so far to control AIDS (Mellors, (1996), Nat. Med. 2:274-276).
Rapid progress on the structure and activity of HIVPr has taken place since the discovery that it is an aspartic protease (Toh et al. (1985), Nature 315:691-692). These include the identification of the HIVPr genome, expression and purification of recombinant enzyme, total chemical synthesis (Schneider and Kent (1988), Cell 54:363-368), crystal structure of HIV-1 protease (Wlodawer et al. (1989), Science 245:616-621; Navia et al. (1989), Nature 337:615-620; Lapatto et al. (1989), Nature 342:299-302) and HIV-2 protease (Mulichak and Watenpaugh (1993), J. Biol. Chem. 268:13103-13109); Tong et al. (1993), Proc. Natl. Acad. Sci. USA 90:8387-8391; Chen and Kuo (1994), J. Biol. Chem. 270:21433-21436) and many enzymic property and inhibition studies. These results are well documented in reviews (Debouck and Metcalf (1990), Drug Devel. Res. 21:1-17; Tomaselli et al. (1991), Chimicaoggi-Chemistry Today 9:6-27; Graves (1991), Adv. Exp. Med. Biol. 306:395-405; Wlodawer and Erickson (1993), Science 245:616-621); and a book (Kuo (1994), Methods in Enzymology Vol. 241).
The active HIVPr is a homodimer of 99-residue monomers. The active-site cleft is located between two monomers with two Asp25 residues forming the catalytic apparatus. The active-site cleft is covered by two flaps and can accommodate eight substrate residues. The specificity of the enzyme is somewhat broad (Poorman et al. (1991), J. Biol. Chem. 266:14554-14561) which is consistent with the sequence differences of the eight natural processing sites. An unique specificity of HIVPr is the ability to cleave an X-Pro bond, which appears to be related to the mobility of the active site of the enzyme (Hong et al. (1998), Protein Sci. 7:300-305). The specificity of the subsite pockets is also influenced by the side chains bound in adjacent pockets (Ridky et al. (1996), J. Biol. Chem. 271:4709-4717).
Each of the four commercial HIVPr inhibitor contains an isostere —CH(OH)—CH2— which mimics the transition state in the catalytic mechanism of aspartic proteases (Marciniszyn et al., 1976), J. Biol. Chem. 251:7088-7094) thereby rendering the tight binding properties of the inhibitor. The position of the isostere, which is equivalent to that of the scissiled bond in the substrate, defines the subsite binding for the inhibitor residues. An example can be seen in a non-commercial inhibitor U-85548 in FIG. 1a. The commercial inhibitor drugs, which require good pharmacokinetic properties and high potency, are typically shorter and have less well defined residue boundaries (FIGS. 1b-d). The interaction of subsite residues in HIVPr with the inhibitors are generally known from the crystal structures of the HIVPr-inhibitor complexes (Wlodawer and Erickson, 1993). The ability of HIVPr inhibitors to suppress HIV replication has been demonstrated in tissue culture and in clinical trials (Wei et al., 1995; Ho et al., 1995). The use of HIVPr inhibitors along with other drugs in combination therapy has offered the best results so far in suppressing HIV propagation in vivo (Mellors, 1996).
The first transition-state analogue of aspartic proteases discovered was pepstatin by Marciniszyn et al. (1976). In this study, the hydroxyethylene group, —CH(OH)—CH2—, was identified to mimic the transition state of catalysis with two carbons in tetrahedral conformation, as contrast to the planar conformation of the peptide bond in the substrate. It was concluded that the potency of pepstatin inhibition was related to the presence of this transition-state mimicry in this (isostere) structure. Because the aspartic proteases share common active-site structure and catalytic mechanism, the transition-state isosteres are applicable to all enzymes of this family. This principle was used later to design inhibitors for renin (Szelke, 1985; Boger, 1985) and HIVPr inhibitors (Tomaselli et al., 1991). Other types of isosteres were later designed and shown to be effective in aspartic protease inhibitors. These include, in addition to hydroxyethylene, dihydroxyethylene [—CH(OH)—CH(OH)—], hydroxyethylamine [—CH(OH)—CH2—NH—], phosphinate [—PO(OH)—CH2—] and reduced amide [—CH2—NH—] (Reviewed by Vacca, 1994). In all cases, including the commercial HIVPr inhibitor drugs, a single transition-state isostere is used in an inhibitor since it minics a substrate peptide with a single hydrolysis site.
The development of resistance to HIVPr inhibitors by the mutation of the HIVPr gene has been clearly demonstrated in in vitro experiments (reviews: Mellors et al. (1994), Nat. Med. 2:760-765; Ridky and Leis (1995), J. Biol. Chem. 271:4709-4717) and in clinical trials (Wei et al. (1995), Nature 373:117-122; Ho et al. (1995), Nature 373:123-126; Jacobsen et al. (1996), J. Infect. Diseases 173:1279-1389; Condra et al. (1996), J. Viol. 70:8270-8276; Molla et al. (1996) Nat. Med. 2:760-765). The in vitro selection of resistant mutants typically takes many passages of HIV in cell culture with increasing inhibitor concentrations for each passage. In patients, the resistance occurs within weeks, owing to the fast replication of virus and fast turnover of the CD4+ T-cells (Coffin, (1995), Science 267:483-489).
Comment: U-85548 is an HIV protease inhibitor, but it is not marketed as anti-HIV drug. We use U-85548 here to illustrate the principle of HIV Protease inhibitor design. The other 3 (1b, 1c, 1d) are drugs.